JP2009539778A - Insulin oligomer derivative - Google Patents

Abstract

The present invention provides oligomers of phosphorylated insulin and formulations thereof.
The oligomeric derivatives of this patent showed pharmacokinetic properties that were significantly improved over natural or intermediate acting or basal insulins (eg, NPH, Lantus, Detemil). And it showed 4 times higher treatment index and 4 times lower risk of hypoglycemia. The present invention provides the benefits of long-lasting blood glucose reduction and combines the benefits of reducing the risk of hypoglycemia. The above is not a characteristic of any currently known insulin, or basal insulin, or intermediate acting insulin. In further embodiments of the present invention, oligomeric phosphorylated insulin is administered for all routes of administration, including inhalation, buccal (oral), subcutaneous injection, infusion, or other technically proven routes of insulin administration. Is suitable. The present invention additionally provides the benefits of a long acting formulation for inhalation between meals and at bedtime.
[Selection] Figure 1A

Description

  The present invention relates to medicine and in particular to the treatment of diabetes and hyperglycemia.

  Diabetes is a debilitating disease that affects more than 5% of the world's population. Approximately 15 million people in the United States have diabetes. Diabetes symptoms include hyperglycemia. Diabetic patients exhibit reduced sensitivity, production, and / or release with respect to insulin. Diabetes is classified as type 1 or insulin-dependent diabetes (IDDM) and type 2 or non-insulin-dependent diabetes (NIDDM). Type 1 diabetes stops the pancreas from producing insulin, affecting 10% of all diabetic patients. It often begins in childhood and is also known as juvenile diabetes. In more common type 2 diabetes or adult type diabetes, insulin production is maintained, but the secretory effect in response to a meal is delayed and / or decreased. And diabetic tissue is often less sensitive to the effects of insulin.

  In both types, decreased insulin response or low levels of insulin result in chronic high levels of blood glucose. And it gradually changes normal bodily physiology, increasing the risk of microvascular and macrovascular sequelae including the kidneys, heart, retina, nerves and cardiovascular complications. Such complications arise mainly from insufficient control of blood glucose and are a serious health problem. Results from a study on diabetes control and complications (DCCT) demonstrate that improved glucose control reduces the incidence of diabetic complications [1].

  However, achieving such control requires insulin therapy, which reflects the type of endogenous insulin secretion seen in normal people. In healthy people, the daily insulin secretion action varies as needed: (A) In the dietary absorption phase, insulin is increased up to 10 times to process dietary glucose, amino acids and other foods, and (b) lower, relatively constant in the postabsorption phase, Sustained amounts of insulin (basal supply) release occur to balance liver glucose production and peripheral nerve glucose uptake. And thereby maintaining standard blood glucose. Effective therapy therefore requires the use of a combination of two types of exogenous insulin, a fast acting dietary insulin and a longer acting or intermediate acting basic insulin.

  HbAlC is a commonly used indicator of blood glucose. The 2006 American Diabetes Association (ADA) treatment goals and recommendations include glycated hemoglobin (HbAlC) as close to the standard (<6%) as possible without significant hypoglycemia, and a target pre-breakdown glucose of 130 mg / dL or less Is included. These ADA recommendations are based on findings that prove that abnormally high glucose is linked to disease complications. In a number of large multicenter Phase 3 clinical trials, to date, no ADA treatment guidelines for mean fasting blood glucose (FBG) of 130 mg / dL or less have been achieved without insulin or a combination thereof. Furthermore, the usual best average study population HbAlCs observed in large trials is not less than 6%, but rather in the range of 7-8%.

  In phase 3 clinical trials, improved “basal” insulin, Lantus & Demir, circumvented the ADA clinical recommendation more fully and achieved a 145 mg% FBG significantly above normal (<100 mg / dL). Unfortunately, existing insulins like NPH or basal insulins like Lantus, or Dememir alone or in combination with fast-acting dietary insulin like lispro, control treatment guidelines for fasting glucose on a daily basis Can not.

  Clearly, there remains much clinical need for improved basal insulin prescriptions that tightly control between-meal glucose.

  As will be appreciated by those skilled in the art, long acting insulin formulations are obtained by making standard insulin as a microcrystalline suspension for subcutaneous injection. Examples of commercially available basic insulin preparations include NPH (Neutral Protamine Hagedorn) insulin, protamine zinc insulin (PZI) and Ultralente (UL).

  NPH insulin is the most widely used insulin preparation, accounting for 50 to 70 percent of the insulin used worldwide until very recently. It is a suspension of a microcrystalline complex of insulin, zinc, protamine and one or more phenolic preservatives. NPH-like preparations of monomeric insulin analogues, LysB298, ProB29-human insulin analogues are known in the art [Patent Document 1: De Felippis, M. et al. R. , Issue date May 5, 1998].

  Currently available treatments with NPH insulin preparations do not provide the ideal “flat” absorption distribution necessary to maintain optimal fasting blood glucose for long periods between meals. Therefore, treatment of NPH insulin can result in undesirably high blood insulin levels. This may cause life-threatening hypoglycemia.

  In addition to not being able to give a flat absorption distribution profile that is ideal, the duration of action of NPH insulin is also poor. Often there is no next morning. In particular, the main problems of NPH therapy are the “mania phenomenon” and hyperglycemia. It is due to losing effective glucose control before waking up. Ultralente is a crystalline preparation of insulin that contains zinc at a higher concentration than NPH, and contains neither protamine nor phenolic preservatives. Human ultralente preparations work longer, but their action is actually variable and not constant.

  Fatty acid insulin has been described [Non Patent Literature 2]. Insulin detemir (Novo Nordisk) was recently released. The fatty acid chains used in fatty acid-acylated insulins are generally longer than approximately 10 carbon atoms, and chain lengths of 14 and 16 carbon atoms are optimal for binding to serum albumin and prolonging action. Fatty acid-acylated insulins such as detemir will dissolve at normal therapeutic concentrations of insulin, but will thereby bind to albumin in the blood, which provides long-term effects. However, the time effects of these formulations are not flat enough to give normal and fundamental control.

Insulin lantus is an insulin analogue with basic properties. It is insoluble at physiological pH (similar to detemir), but prevents some of the absorption variability observed in insulin suspensions by being soluble at the pH at which it was made. Precipitation occurs in the injection solution, resulting in long lasting absorption. Moderately flat absorption can be obtained with this insulin, but when this insulin is tested in a large clinical trial, it is unacceptable due to daily fluctuations in absorption along with the steep dose-response curve of insulin. It is not a robust enough therapy to achieve the ADA glucose goal without producing hypoglycemia.

Various phosphorylated insulins (P insulins) have been disclosed. A method using phosphoric acid (Non-patent Document 3), a method using phosphoric acid / POCL ₃ in a non-aqueous organic solvent using a binder (Patent Documents 2 and 3: Cerami A. et al), or phosphorami Insulin is phosphorylated by the method used together with the date (Non-patent Document 4). Phosphorylated insulin created by Ferrel et al. And Rathlev and Rosenberg was part of a study designed to further understand the process of phosphorylation. And especially to increase the learning of how it relates to biological systems. The clinical effect of this phosphorylated insulin was not measured or observed. In the above prior art, the specific amino acid or its site where insulin was phosphorylated was not determined. Therefore, the specific molecular structure was not revealed and patented. Furthermore, none of the above prior art has published insulin using altered pharmacodynamics. In contrast, the present invention describes insulin using a flattened glucose insulin dose response curve.

  The patent granted to Cerami et al. (Cited above) includes sulfonated and phosphorylated insulins with the advantages of reduced polymerization when stored for long periods in an insulin delivery system.

  These insulins claim an effect that does not cause obstruction of the insulin pump. And therefore, they must have better control of blood glucose. The Cerami et al. Patent emphasizes that improvements in the process are achieved by performing phosphorylation in a non-aqueous solvent. Thus, they teach that P insulin is likely to produce hypoglycemia (mouse seizure analysis). Therefore, contrary to the results described here, the above Cerami et al. Insulin is different in that it does not show a reduced likelihood of hypoglycemia. In particular, it must be concluded for this that the chemical identity of P insulin described by Cerami et al. Is significantly different from the present invention. Cerami et al. Were unable to determine the scientific identity of a heterogeneous mixture of phosphorylated insulin produced by their patent (with a range of isoelectric moieties). And chemical comparisons by those familiar with the technology are not possible without such.

As demonstrated by Non-Patent Document 5, insulin is also phosphorylated with POCl ₃ along with excess pyridine. This reference reveals that insulin may be phosphorylated in an anhydrous medium, essentially without modification of its hypoglycemic effect. The chemical composition was not revealed and not determined. Also, no beneficial effects were described or evaluated in glucose control.

  The author's patent (Patent Documents 4 and 5: Lowheed, WD) was recognized on the prior art cited here and by Albisser (Patent Document 6). Patent document 6 by Albisser differs from the present invention in that the monomeric P insulin is described as follows ["characteristically the modified product intact monoclonal insulin" (p. 4, 1.23-24) ]]. In contrast, the present invention discusses insulin oligomers. Excellent pharmacological action has been obtained from oligomers. Although Albisser reveals amino acid sequence results, it is not specific and one or more of the three possible serine residues of insulin are phosphorylated as O-phosphate.

  The phosphorylated insulin chemical composition cannot be determined from this sequence data.

  The Lockheed patent (Patent Documents 4 and 5) describes a monomeric phosphorylated insulin with unique properties. It shows good results in reducing hypoglycemia, and the rapid dosing response of insulin is flattened 3-4 times.

  By reducing the risk of hypoglycemia, insulin is more aggressively given, resulting in improved glucose control. The present invention here differs from the inventors' previous work in that it describes the oligomeric phosphorylated insulin has a surprising additional utility of significantly increasing its serum half-life. This property provides basal insulin with the prolongation required to control glucose between meals.

  Insulin administration to the lung can be accomplished with aqueous solutions or powders. Formulas for such dry insulin powders, aerosols or liquid inhalants have been extensively analyzed. Examples are given below (Patent Documents 8 and 9, Non-Patent Documents 7 and 8).

  For example, as shown in Non-Patent Document 9, many have shown that glucose control is provided to patients with type 1 and type 2 insulin-dependent diabetes. The anhydrous powder insulin formulation described by Patton et al. Overcomes the problem of formulation instability.

  Absorption is increased by the use of enhancers as discussed by Sayani et al. (Non-Patent Document 10) or by utilizing a prescription without zinc (Patent Document 10).

  As described by Patton et al. (Inhalation Therapy System, US Pat. No. 6,099,059), anhydrous powdered insulin is dispersed into a gas stream and is taken up to form an aerosol. Then weigh and dispense. Steiner SS et al., US Pat. Such particles have a large fluted surface area that enhances lung absorption with zinc removal.

  Mannitol combined with various buffers may be used to increase the stability of inhalable formulations, as described in Platz et al. Small particle size, 0.2-5 mu. The order of m is optimal for deep lung penetration.

  Insulin to the lung, which has a long-term effect, was also recently described in the following US patents (patent documents 14-17). As tested in animal models, they have been shown to have long-term effects in preclinical studies of glucose control between meals. The effectiveness in humans has not yet been demonstrated. While these lung prescriptions show promise for providing basic lung delivery, as shown in this application, long-term effects combined with results that reduce the effects of hypoglycemia Not shown.

  The American Diabetes Association Expert Advisory Committee on Diabetes has defined the need for insulin that reduces the likelihood of hypoglycemia and has prolonged action. [Non-Patent Document 11].

  The long serum half-life of oligomeric P insulin compounds meets the need to provide a long-acting formulation for treating diabetes while reducing the incidence of hypoglycemia. Treatment may be by any defined treatment regimen, such as administration using a needle, pen, inhalation system, mouth-blowing, needleless injection system or insulin pump.

US Pat. No. 5,747,642 US Pat. No. 4,534,894 US Pat. No. 4,705,845 US Pat. No. 5,453,417 PCT / CA92 / 00082 US Pat. No. 5,242,900 US Pat. No. 4,639,333 (Obermeier R. et al., Filled January 27, 1987) US Pat. No. 5,997,848, Patton, et al. , U.S. Pat. No. 5,506,203, Platz et al. Inhale Therapeutic Systems, PCT WO 96/32149. Boederke, P.M. : US Pat. No. 6,960,561 WO95 / 24183 U.S. Pat. WO96 / 32149 US Pat. No. 6,465,426, Brader ML, issue date October 15, 2002 US Pat. No. 6,808,076, Patton JS et al, issued January 4, 2005. US Pat. No. 6310038, Havelund S, date October 30, 2001 U.S. Pat. No. 6,335,316, Hughs BL et al, issue date January 1, 2002 Chance, et al. U.S. Pat. No. 5,514,646, issued May 7, 1996. EPO Publication No. 383472, Feb. 7, 1996 Brange, J. et al. J. et al. V. , Et al. EPO publication number 214826, Mar. 18, 1987 Platz, et al. , International Publication WO 96/32149, Publication Date Oct. 17, 1996 Patton, et al. , International Publication WO95 / 24183, Release Date Sep. 14, 1995 U.S. Pat. No. 5,622,166, issued on Apr. 22, 1997 Meikalski, et al. U.S. Pat. No. 5,577,497, Nov. 26, 1996 Meikalski, et al. U.S. Pat. No. 5,492,112, issued Feb. 20, 1996 Williams, et al. U.S. Pat. No. 5,327,883, issued on JuI. 12, 1994 Goodman, et al. U.S. Pat. No. 5,404,871, issued on Apr. 11, 1995 Rubsamen, et al. U.S. Pat. No. 5,672,581, issued Sep. 30, 1997 Gonda, et al. U.S. Pat. No. 5,743,250, date of issue Apr. 28, 1998 Rubsamen, US Pat. No. 5,419,315, issued May 30, 199 Rubsamen, et al. U.S. Pat. No. 5,558,085, issue date Sep. 24, 1996 Johnson, et al. U.S. Pat. No. 5,654,007, date of issue Aug. 5, 1997 Gonda, et al. , International Publication WO 98/33480, Publication Date Aug. 6, 1998 Rubsamen, US Pat. No. 5,364,833, issued Nov. 15, 1994 Laube, et al. , U.S. Pat. 14, 1994 Eljamal, et al. U.S. Pat. No. 5780014, issued on JuI. 14, 1998 Backstrom, et al. U.S. Pat. No. 5,658,878, date of issue Aug. 19, 1997 Backstrom, et al. U.S. Pat. No. 5,518,998, issued May 21, 1996. Backstrom, et al. U.S. Pat. No. 5,506,203, date of issue Apr. 9, 1996 Meezan, et al. U.S. Pat. No. 5,661,130, date of issue Aug. 26, 1997 Schultz, et al. U.S. Pat. No. 5,654,051, issued Jul. 8, 1997 Rubsamen, US Pat. No. 5,364,838, issue date Nov. 15, 1994 Rubsamen, US Pat. No. 5,672,581, issued Sep. 30, 1997 Williams, US Pat. No. 5,277,195, date of publication Jan. 11, 1994. Markussen, et al, U.S. Pat. No. 4,916,212 filled May 29, 1985; date of publication April 10, 1990. U.S. Pat. No. 4,559,300 (Kovacevic, S. et al. Issued December 17, 1985, Di Marchi R., filled August 1, 1983) U.S. Pat. No. 4,661,078, Di Marchi R .; , Filled October 7, 1986

The DCCT Research Group. New. Engl. J. et al. Med. 329, 977-986 (1993) Whittingham, J.M. L. , Et al. [Biochemistry 36: 2826-2831 (1997) Ferrel R.M. E. et al. , Journal of the American Chemical Society, 70, 2107-7, 1948. Rathlev, V.M. and Rosenberg T .; , Archives of Biochemistry and Biophysics, 65, 319-339, 1956 Royal Z et al., Chemical Abstracts, Vol. 68, 1968, (Columbus, Ohio, United States) R. E. Chance et al: Diabetes Care 4, 147 (1982) Inhale Therapeutic Systems, Inc: Niven, Crit. Rev. Ther. Drug Carrier Sys, 12 (2 & 3): 151-231 (1995) Sayani et al. , Crit. Rev. Ther. Drug Carrier Sys, 13 (1 & 2): 85-184 (1996). Patton, et al. , Adv. Drug Delivery Reviews, 1, 35 (2-3), 235-247 (1999) Crit. Rev. Ther. Drug Carrier Sys, 13 (1 & 2): 85-184 (1996). “More effect is needed to develop treatments who reduce the risk of hypoglycemia”, 1997 FDA policy statement in diabetes “Current Methods of Phosphorylation of Biological Moleculares”, Slotin LA, Synthesis: 737-752 (1977).

  This application is filed on June 8, 2006, in United States prior patent application no. Claim the benefit of the 60 / 811,766 priority date. That is all incorporated here.

  An improved insulin composition is provided that reduces the effects of hypoglycemia and has prolonged action therapy for glucose control between meals. Surprisingly, the inventor has defined sequence ID no. 7 & 8 P insulin was found to cause oligomerization. The oligomerization has the following beneficial properties:

  1. Prolong absorption from the subcutaneous tissue and further increase the serum half-life of the hormone. This has the effect of further reducing the risk of hypoglycemia compared to the inventor's previous patent. 2. Significantly extend the duration of action. Such pharmacodynamic properties enhance glycemic control during the meal.

  The present invention thus relates to the formulation of large amounts of drugs and P insulin oligomers. The P insulin, importantly, has the advantage of a sustained duration of action and reduces the effects of hypoglycemia. They are described by the sequence of oligomers of the formula [X] n or more specifically as follows:

XX ID NO. 1 or
XX ID No. 2 or
X-X-X ID No. 3 or
X-X-X-X ID No. 4 or
X-X-X-X-X ID No. 5 or
[X-] ⁿ ID No. 6 ("X" is repeated n times)
“X” is the following sequence ID No. 7 or ID No. Limited to 8.

  Sequence ID NO. 7

“—P” is defined to indicate an O-phosphorylated amino acid residue. “R” means an amino acid residue that can be further O-phosphorylated. Lower case “s” means sulfur.

Or
“X” is the following sequence ID NO. 8 is shown;

  “-P” is defined to indicate an O-phosphorylated amino acid residue. The lower case “s” means sulfur.

The manufacturing method of the present invention will now be described with reference to the following accompanying drawings.
Size exclusion chromatography using Q Sepharose in 99/1 50 mM ammonium phosphate, pH 9.0 / acetonitrile. a. Oligomers (tetramers, hexamers and octamers) b. Molecular weight standard. SDS polyacrylamide gel electrophoresis of phosphorylated insulin produced according to US Pat. Only phosphorylated insulin monomer with a molecular weight of 6000 da is shown. Mass spectrometry of P insulin oligomers. 2a. MALDI-MS of P insulin oligomers. During MALDI-MS, higher oligomers are cleaved to generate p insulin monomer (5808da), dimer (11,616) and trimer (17,424da). P-insulin was reduced and alkylated to break the disulfide bond, and the A and B chains were disconnected. Masses of both A chain and B chain were observed by MALDI-MS. The results revealed that there are two phosphate sites in the A chain and four phosphate sites in the B chain. The phosphorylation site was positioned in the A chain for Ser9 and Tyr6 by MS / MS (FIG. 2B). And to locate the remaining phosphorylation sites, reduced and alkylated P insulin was digested with trypsin overnight. The resulting fragment was shown on slide # 15-17. Two phosphorylation sites were determined in insulin B chain Tyr26 and Thr27 (FIG. 2C). Intravenous injection of human insulin and P insulin into a pancreatic diabetic beagle dog. A. Rapid decrease in blood levels of human insulin. Hypoglycemia at 15 minutes, rapid recovery of glycemia with short serum half-life after injection. B. P insulin oligomers at various doses. Prolonged duration of action (long serum half-life), showing normal glucose maintenance without hyperglycemia at all doses. Hypoglycemia was not observed until the 8 times baseline dose or “dose to maintain glucose” was achieved. Subcutaneous injection of human insulin and P insulin oligomers into diabetic beagle dogs. Shows that the duration of action is prolonged compared to unmodified insulin. Dose response curve of human & porcine & P insulin oligomers in L6 murine myocytes. The cells were incubated for 60 minutes with different concentrations of each insulin in each experiment. The intake of 2-deoxyglucose was then measured. P insulin oligomers did not show a flat dose response at all concentrations investigated. Surprisingly, P insulin oligomers showed a maximal response that was significantly lower than porcine or human insulin. Glucose response of phosphorylated insulin hexamer 40 mg sprag-dolly rat. Rats were injected Oh and glucose levels were measured for 12 h. As shown, glucose levels dropped to 2 h after taking and were reduced to 11 h after taking, after which they returned to baseline or pre-dosing levels.

  This indicates that the P insulin oligomers are less likely to cause hypoglycemia even at overdose concentrations.

  The following abbreviations are used to represent amino acids.

Insulin oligomers comprising one of the following sequences are provided.
XX (ID No. 1),
X-X-X (ID No. 2),
XX-X-X (ID No. 3),
XX-XX-X (ID No. 4),
X-X-X-X-X (ID No. 5), and
[X-] ⁿ (ID No. 6), X is repeated n times.
X is one of the following:

  (A) Sequence ID No. 7. Insulin as defined by 7 or a pharmaceutically acceptable salt thereof. Sequence ID No. 7 is shown below.

  P represents an O-phosphorylated amino acid residue.

R is an optionally O-phosphorylated amino acid residue and the lowercase “s” is sulfur. Or
(B) Sequence ID NO. Insulin as defined by 8 or a pharmaceutically acceptable salt thereof. Sequence ID NO. 8 is shown below.

  P represents an O-phosphorylated amino acid residue, and the lowercase “s” is sulfur.

  Sequence ID No. Insulin defined by 7 or 8 can be an insulin analogue.

  As another example, one or more residues at positions R1, R2, R3 or R4 need not be phosphorylated.

  A method for treating diabetes is provided to a patient in need thereof. The method includes administering an effective dose of an insulin oligomer, as defined above, or the like.

  Instead, it includes administering an effective dose of an insulin oligomer or the like by oral absorption, oral, or subcutaneous injection, by inhalation, a combination of subcutaneous infusions, or either.

  A method for treating hyperglycemia is also provided to patients in need thereof. The method consists of orally administering an effective dose of an insulin oligomer, as defined above, or the like.

  Also provided is a composition comprising an insulin oligomer consisting of one of the above sequences as defined above. The composition further comprises a pharmaceutically suitable diluent, excipient, or carrier thereof. Compositions containing insulin oligomers are used in the treatment of hyperglycemic diabetes.

In the preparation of an insulin oligomer consisting of one of the following sequences: 7, Sequence ID No. Use of insulin as defined by 8 or a pharmaceutically acceptable salt thereof.
XX (ID No. 1),
X-X-X (ID No. 2),
XX-X-X (ID No. 3),
XX-XX-X (ID No. 4),
X-X-X-X-X (ID No. 5), and
[X-] ⁿ (ID No. 6), X is repeated n times.
Sequence ID No. 7 and sequence ID no. X, which is any one of 8 insulins, is also provided.

Administration of phosphorylated insulin oligomers can be via any route known to be effective by medical personnel. Here, parenteral is defined as “administration by a route other than through the gastrointestinal tract”. Parenteral routes for practicing the formulations of the present invention include subcutaneous, pulmonary, arterial, peritoneal, muscle, nasal, intravenous, and oral routes.
The formulations of the present invention can utilize a catheter, or an infusion system, or a needle that is introduced under the catheter or skin. Or conversely, an inhaler for administration in the lungs or buccal cavity can be utilized.

  Furthermore, the P insulin oligomers provided by the present invention can be implemented by any other means recognized in the art for parenteral administration. The P insulin oligomers of the present invention can also be formulated as dry powders or aerosols for absorption into the lungs, nasal cavity, sublingual space or buccal mucosa.

  The amount of P insulin oligomers that is performed to control glucose depends on many factors. This includes, without limitation, the route of administration, the efficacy of the formulation, weight and age, type of diabetes, the bioavailability of the route of administration and the route, the in vivo half-life of the P insulin performed, and the formulation. It is known that the bioavailability of insulin can be reduced via various routes including nasal, buccal and pulmonary therapy. And the dose needs to be adjusted more and adjusted individually by the doctor or other medical personnel trained in these routes. Continuous administration is achieved by any physician skilled in the art for adjusting the insulin dose. Intermittent administration is accomplished by those skilled in the art as well. And doses that need to be controlled between medications can be taken into account.

  Insulin and Pinsulin of the present invention can be made by any of a variety of recognized protein / peptide synthesis techniques. These include recombinant DNA methods, semi-synthetic methods, solid phase or solution methods, and methods known in particular to incorporate amino acids or amino acid phosphates into peptides and to create insulin analogues (patents) Literatures 18 to 20, Non-Patent Literatures 12 and 7). These patents provide sufficient detail to allow those skilled in the art to prepare the P insulin of the present invention for various peptides, amino acids, amino acid phosphates, insulin or insulin analogs. The preparation is publicly available. Appropriate kinases can also be used on the phosphorylated tyrosine, serine, and threonine residues of insulin to produce the products of the invention.

  In accordance with the present invention, p-insulin oligomers are used in any of a variety of inhalation devices and are known in the art for the preparation of pulmonary insulin formulations, as exemplified below. It can be prepared by a method (Patent Documents 21 to 44). The entire publication of each of the publications listed above is expressly included herein by reference.

  The aqueous solution formulation of the present invention can be prepared by dissolving P insulin at H4.0-8.0. Although dissolution is possible at pH 9.0 and above, exposure should be short. Alternatively, the temperature must be lowered to limit the amino acid hydrolysis of insulin that is known to occur. Within the pH range that meets the above conditions, dissolution and preparation is not limited and includes any citrate, phosphate, acetate, sodium chloride, hydrochloric acid, phosphoric acid, sulfuric acid, trishydroxyaminomethane and glycerin. Physiologically acceptable non-toxic salts, isotonic agents and buffers can be used.

  It is highly preferred that the formulation be made isotonic to humans, as the method for doing so will be apparent to those skilled in the art.

  The nature and concentration of the additive to the formulation, similar addition order drug concentration, pH, temperature during adjustment are optimized for the formulation and the route of administration is also contemplated.

  The term “P insulin” has the sequence ID No. All phosphorylated insulin oligomers from 1 to 6 are meant.

  The term “phenol preservative” means any of m-cresol, phenol, and methylparaben. In order to maintain the sterility of the insulin formulations of the present invention when they are used in vials, cartridges, syringes or other devices where multiple contacts and / or multiple uses are required, a phenolic preservative is used. Needed. Such preservatives are not needed in single uses such as cartridges, envelopes, and dry powder packages as used in inhalation and aerosol devices.

  The term “insulin analogue” means a protein having an A chain and a B chain having substantially the same amino acid sequence as the A and B chains of human insulin, respectively. However, by having one or more amino acid additions and / or one or more amino acid substitutions, one or more amino acid termini that do not destroy the insulin activity of the insulin analog, the A and B chains of human insulin Is different.

  The term “treating” as used herein refers to the medical treatment of patients with diabetes or hyperglycemia. And the insulin administration has the purpose of reducing glucose, reducing the symptoms of hyperglycemia and the metabolic sequelae that come from such. “Treatment” is intended to maintain or restore good blood sugar, prevent hyperglycemia, reduce hyperglycemia, and otherwise prevent, ameliorate, or reduce diabetes complications, This includes administering to a diabetic patient a dose of the present invention in either solid phase or liquid form.

  The following test examples are only provided to further illustrate and describe the present invention. The scope of the invention is not limited to the following examples.

Test example 1
Insulin prepared by the method described in U.S. Patent No. 6,057,037 was dialyzed to remove all zinc to optimize the post-reaction composition of the appropriate monomer precursor for the P insulin oligomer. . Phosphorylation at −2 to + 2 ° C. with a minimum starting concentration of 50 mM potassium phosphate for 60 minutes and excess POCl ₃ (360 μL) gives a phosphate concentration at the solubility limit. . This produced a high yield of oligomeric insulin phosphate. Oligomers were separated by size exclusion chromatography on a Zorbax 250 column or similar column with Q Sepharose using 50 mM ammonium phosphate buffer (pH 9.0) supplemented with 1% (v / v) acetonitrile. By this method as shown in FIGS. 1a and 2a, dimers, trimers and hexamers of phosphorylated insulin can be prepared. Insulin monomer retention time is shown in FIG. 1b for comparison. Samples were handled together by HPLC analysis of the appropriate molar weight standard to determine the molar weight. FIG. 1c shows the P insulin monomer produced by Patent Document 4 (Lockheed, WD) as a comparison. SDS polyacrylamide gel electrophoresis showed the absence of MW 8,000 Da P insulin monomer and insulin oligomer.

Test example 2
The human insulin precursor was prepared according to Patent Document 46 and Patent Document 47, and was converted to human insulin by the method described in Patent Document 7.

Phosphorylation at −2 to 0 ° C. used a minimum starting concentration of 50 mM potassium phosphate and excess POCl ₃ (360 μL) in a 60 minute reaction. The reaction was quenched with 5 times the amount of non-ionized ice. A high yield of oligomeric phosphorylated insulin was obtained.

  Oligomers were separated by size exclusion chromatography on a Zorbax 250 column using 50 mM ammonium phosphate buffer (pH 9.0) supplemented with 1% (v / v) acetonitrile. By this method, dimers, trimers and hexamers of phosphorylated insulin could be prepared.

Test example 3
The P insulin oligomer formulation was prepared as follows. By size exclusion chromatography, 40 mg of P insulin tetramer isolated as above was added to 9 mL of 50 mmol sodium chloride, 15 mmol sodium phosphate. Glycerin was added to make the solution isotonic in the final amount. The pH was adjusted to 7.4 with 1N NaOH and / or 1N HCl. M-cresol / phenol was added to an equimolar concentration of 0.125% (based on a final volume of 10 milliliters). The volume was made up to 10 mL and was sterile filtered and stored at 4 ° C.

Test example 4
Mass Spectrometer The MALDI and tandem mass spectrometer was performed on the P insulin prepared in Example 1 while keeping the reaction temperature at 0 ° C. The results are shown in FIGS. 2A-2D. As demonstrated by isoelectric focusing using isoelectric focusing, P insulin monomers, dimers, trimers, tetramers and hexamers each contained 5 phosphate groups. By sulfonic acid degradation to obtain A & B chains, followed by MALDI and tandem mass spectrometry, the X of these oligomers is sequence SEQ ID No. 7 (FIGS. 2B-2D).

Test Example 5
Unmodified human insulin or sequence ID no. Seven human insulin P-insulin oligomers were injected intravenously into pancreatic diabetic dogs (FIG. 3). As shown in FIG. 3a, unmodified insulin exhibited a sharp drop in blood glucose levels, marked hypoglycemia and a very short duration of action (20 minutes when given IV).

  With significant contrast, oligomers of P insulin (Figure 3b) showed no or minimal decrease in blood glucose levels (except very high doses), no hypoglycemia at all doses, and unmodified insulin The effect was 10 times longer.

Test Example 6
Sequence ID NO. Eight human insulin P-insulin oligomers were injected intravenously into Fisher 344 mice (n = 30). With injections up to 2,000 times the expected human dose, only very high doses (approximately 0.15 mg / kg body weight) induced hypoglycemia. And no death or convulsions were induced.

Test Example 7
With modified insulin or 3: 1 M: M protamine / insulin, SEQ ID NO. The formulation of Example 3 containing 8 P insulin oligomers was injected intravenously into a diabetic dog. The long-term glucose inhibition of P insulin oligomers is shown in FIG.

Test Example 8
Sequence ID No. Seven P insulin oligomers were tested in L6 murine muscle cell cultures for potential glucose uptake. P insulin oligomers caused the maximal stimulation of deoxyglucose uptake by half that of human insulin. The dose response was flat for the entire dose range in murine muscle cells (FIG. 5).

Test Example 9
Via the pulmonary route, 6 sprag-dolly mice (BW 250-360 grams) were dosed with 4 mg of phosphorylated insulin oligomer (in the form of a hexamer). The animals are allowed to take the drug by intratracheal administration using the Penncentury intrachemical Aerosolizer (Microsprayer). The dose was 100 μL / animal.

  The dosing concentration for the study was prepared by diluting 30 mg / mL phosphorylated insulin hexamer in isotonic, phosphate buffered standard saline, pH 7.4.

  An antisedan cocktail consisting of atibamezol hydrochloride (1 mg / mL, 1 mg / kg) and physiological saline were administered by intravenous injection of 1 mL / kg in a single dose, and the dosing process was achieved. A series of 13 blood samples (approximately 0.1 mL / sample) were collected from the animals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours after each dose.

  For this purpose, each mouse (unanesthetized) was bled at the jugular / tail vein puncture (or other place where it was needed). The samples were then used to measure whole blood glucose levels using the Accu Soft Advantage (trademark: glucose meter) method. A sample for plasma phosphorylated insulin levels was also taken.

  Following pulmonary administration (FIG. 6), blood glucose levels have been moderately suppressed after 2 hours. He remained below the baseline for 11 hours after taking it. By 12 hours the glucose level had returned to the pre-dose level.

  It will be appreciated that other embodiments and examples of the invention will be readily apparent to those skilled in the art since the scope and authority of the invention is defined in the appended claims.

Claims (12)

Insulin oligomer consisting of one of the following sequences:
XX (IDNO.1),
X-X-X (ID NO. 2),
XX-X-X (ID No. 3),
X-X-X-X (ID No. 4),
X-X-X-X-X (ID No. 5), and
[X-] ⁿ (ID NO. 6), X is repeated n times;
X is the sequence ID NO. 7. Insulin defined in 7 or a pharmaceutically acceptable salt thereof. Sequence ID NO. 7 is shown below.

P represents an O-phosphorylated amino acid residue.
R is an amino acid residue optionally O-phosphorylated. And the lowercase “s” is sulfur;
Alternatively, the sequence ID NO. Insulin as defined by 8 or a pharmaceutically acceptable salt thereof. Sequence ID NO. 8 is shown below.

P represents an O-phosphorylated amino acid residue. The lower case “s” is sulfur.   The insulin oligomer according to claim 1, wherein X is an insulin analogue.   2. The insulin oligomer according to claim 1, wherein one or more residues at positions R1, R2, R3 or R4 are not phosphorylated.   A method for treating diabetes in a patient in need thereof, comprising a method of administering an effective dose of the insulin oligomer or the like thereof according to claim 1.   5. The method for treating diabetes according to claim 4, comprising administering an effective dose of the insulin oligomer according to claim 1 or the like by subcutaneous infusion or subcutaneous injection.   5. A method for treating diabetes according to claim 4, comprising administering by inhalation an effective dose of an insulin oligomer according to claim 1 or the like.   5. A method for treating diabetes according to claim 4, comprising administering an effective dose of an insulin oligomer according to claim 1 or the like by absorption through the mouth.   5. A method for treating diabetes according to claim 4 comprising orally administering an effective dose of the insulin oligomer according to claim 1 or the like.   A method for treating hyperglycemia in a patient in need thereof, comprising orally administering an effective dose of the insulin oligomer according to claim 1 or the like. A composition comprising an insulin oligomer consisting of one of the following sequences:
XX (IDNO.1),
X-X-X (ID NO. 2),
XX-X-X (ID No. 3),
X-X-X-X (ID No. 4),
X-X-X-X-X (ID No. 5), and
[X-] ⁿ (ID NO. 6), X is repeated n times;
X is the sequence ID NO. 7. Insulin defined in 7 or a pharmaceutically acceptable salt thereof. Sequence ID NO. 7 is shown below.

P represents an O-phosphorylated amino acid residue R is an optionally O-phosphorylated amino acid residue. And the lowercase “s” is sulfur;
Sequence ID NO. 8. Insulin or a pharmaceutically acceptable salt thereof as defined by 8 and a pharmaceutically acceptable diluent, economy, carrier. Sequence ID NO. 8 is shown below.

P represents an O-phosphorylated amino acid residue. The lower case “s” is sulfur.   Use of a composition comprising an insulin oligomer according to claim 10 in the treatment of diabetes. Use of insulin as defined below:
Insulin oligomer produced comprising one of the following sequences: :
XX (IDNO.1),
X-X-X (ID NO. 2),
XX-X-X (ID No. 3),
X-X-X-X (ID No. 4),
X-X-X-X-X (ID No. 5), and
[X-] ⁿ (ID NO. 6), X is repeated n times;
X is the sequence ID No. 7 and sequence ID No. Any one of 8 insulins.
Sequence ID NO. 7 or a pharmaceutically acceptable salt thereof. Sequence ID NO. 7:

P represents an amino acid residue phosphorylated O.
R is an amino acid residue optionally O-phosphorylated. And the lower case “s” is sulfur. ;
Alternatively, the sequence ID NO. 8 or a pharmaceutically acceptable salt thereof. Sequence ID NO. 8 is shown below.

P represents an O-phosphorylated amino acid residue. The lower case “s” is sulfur.

Images